Microbial Profiling Using Mothur Pipeline
Snakemake workflow
Snakemake workflow
Mothur Reference databases
- Mothur-based SILVA reference files[1].
- Mothur-based RDP reference files[2]. Note: The RDP database is to classify 16S rRNA gene sequences to the genus level.
- ZymoBIOMICS Microbial Community Standard (Cat # D6306)[3]. The ZymoBIOMICS Microbial Community DNA Standard is designed to assess bias, errors and other artifacts after the step of nucleic acid purification.
Mothur classification methods
There are four methods that can be used to profile microbial communities present in a sample. Here we briefly decribe each method:
1.Classify OTUs
- OTUs (Operational Taxonomic Units (OTUs)) are clusters of similar sequences and are commonly accepted as analytical units in microbial profiling when using 16S rRNA gene markers.
2. Classify Phylotypes
- A phylotype in microbiome research is a DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a 16S rRNA gene marker.
3. Classify ASVs
- ASVs Amplicon Sequence Variants (ASVs)in microbiome research is any inferred single DNA sequences recovered from a bioinformatics analysis of 16S rRNA marker genes.
- ASV is typically really a cluster of sequences that are one or two bases apart from each other.
4. Classify Phylogenies
- Microbial phylogenies are from gene sequence homologies. Models of mutation determine the most-likely evolutionary histories.
Preliminary OTU analysis using Mothur
The preliminary analysis (alpha_beta_diversity rule) is
part of the bioinformatics analysis. It includes:
- Creating reads count for each group.
- Subsampling for downstream analysis.
- Rarefaction.
- Computing Alpha diversity metrics.
- Computing Beta diversity metrics.
- Getting sample distances.
- Constructing sample phylip tree.
- Generating ordination matrices including PCoA and NMDS.
…IN PROGRESS…
References
[1]
Mothur-based silva reference files. Retrieved
from https://mothur.org/wiki/silva_reference_files/
[2]
Mothur-based RDP reference files. Retrieved
from https://mothur.org/wiki/rdp_reference_files/
[3]
ZymoBIOMICS microbial community DNA standard
(cat # D6306). Retrieved from https://www.zymoresearch.com/zymobiomics-community-standard
[4]
Buza, T. M., Tonui, T., Stomeo, F., Tiambo, C.,
Katani, R., Schilling, M., … Kapur, V. (2019). iMAP: An integrated
bioinformatics and visualization pipeline for microbiome data analysis.
BMC Bioinformatics, 20. https://doi.org/10.1186/S12859-019-2965-4
Appendix
Project main tree
.
├── LICENSE.md
├── README.md
├── REFER
│  ├── dags
│  ├── data
│  ├── envs
│  ├── images
│  ├── imap-bioinformatics.Rproj
│  ├── index.html
│  ├── render.R
│  ├── report.html
│  ├── resources
│  ├── results
│  ├── rules_dag.sh
│  ├── smk_html_report.sh
│  └── workflow
├── config
│  ├── config.yml
│  ├── pbs-torque
│  ├── samples.tsv
│  ├── slurm
│  └── units.tsv
├── dags
│  ├── rulegraph.png
│  └── rulegraph.svg
├── data
│  ├── README.md
│  ├── metadata
│  ├── mothur
│  ├── reads
│  ├── references
│  └── test
├── images
│  ├── bioinformatics.png
│  ├── bkgd.png
│  └── smkreport
├── index.Rmd
├── library
│  ├── apa.csl
│  ├── export.bib
│  ├── imap.bib
│  └── references.bib
├── report.html
├── results
│  └── project_tree.txt
├── styles.css
├── tree.sh
└── workflow
├── Snakefile
├── envs
├── rules
└── scripts
26 directories, 26 files
Screenshot of interactive snakemake report
The interactive snakemake HTML report can be viewed by opening the
report.htmlusing any compatible browser. You will be able to explore the workflow and the associated statistics. You can close the left bar to get a more expansive display view.
Troubleshooting of FAQs
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